Method for deblocking of labeled oligonucleotides

ABSTRACT

The invention relates to a process for deblocking substantially a blocked, detectably labeled oligonucleotide by contacting the blocked detectably labeled oligonucleotide with an effective amount of a nucleophilic amino compound under conditions that result in substantial deblocking of the oligonucleotide, thereby giving the substantially deblocked oligonucleotide.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. ProvisionalApplication No. 60/135,848, filed May 24, 1999, the contents of whichare entirely incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to processes for the substantialdeprotection or deblocking of labeled oligonucleotides by use of anamino reagent such as ammonia.

2. Related Art

A variety of solid phase oligonucleotide synthesis techniques are knownto those skilled in the art. Such techniques include phosphoramidite,phosphotriester, phosphodiester, phosphite and H-phosphonate methods andthe like, each of which is generally known in the field of molecularbiology. For example, the b-cyanoethyl phosphoramidite method isdescribed in U.S. Pat. No. 4,458,066 issued to Caruthers, et al.,entitled “Process for Preparing Polynucleotides,” which is incorporatedherein by reference.

The phosphoramidite based synthesis of oligonucleotides requires theprotection of the exocyclic amino groups. The most commonly usedprotecting groups for this purpose are benzoyl for the 6-amino ofadenine and 4-amino of cytosine and isobutyroyl for 2-amino of guanine.Oligonucleotides are synthesized on solid support using nucleosidephosphoramidites where the amino groups are protected as shown below.

After the synthesis is completed the oligonucleotide is cleaved from thesupport and these protecting groups are removed by hydrolysis at hightemperatures using concentrated ammonium hydroxide. After hydrolysis,the ammonium hydroxide has to be evaporated in order to obtain thedesired oligonucleotide.

The use of hot concentrated ammonium hydroxide for the removal of theseprotecting groups has restricted the modified bases that can be used tothose that can withstand these harsh conditions. For modified oligossuch as dye containing oligos, heating in ammonium hydroxide can not beused since the dyes are not stable under these conditions. In general,dye labeled oligos arc deprotected by treatment with ammonium hydroxideat room temperature for over 24 hours or require special reagents forthis purpose. See U.S. Pat. No. 4,965,349. Alternatively, oligos mayprepared using phosphoramidite having easily removable protecting groupswhich do not require the use of hot concentrated ammonium hydroxide. SeeBoal, J. H. et al., Nucl. Acids Res. 24:3115-3117 (1996).

The standard method where deprotection with concentrated ammoniumhydroxide is done at reduced temperature results in incompletedeprotection and over all low quality of dye labeled oligonucleotides.Often this requires tedious purification which results in low yield.

U.S. Pat. No. 4,965,349 describes a method of hydrolyzing base-labilelinking groups between a solid phase support and oligonucleotides with areagent comprising a lower alcohol, water and a non-nucleophilichindered alkylamine.

According to this patent, this cleavage reagent preserves thefluorescent characteristics of rhodamine dyes during cleavage from thesolid support.

U.S. Pat. No. 5,514,789 describes a method for the cleavage anddeprotection of newly synthesized oligonucleotides from solid supportswith a gaseous cleavage/deprotection reagent such as gaseous ammonia,ammonium hydroxide vapors, and methylamine.

U.S. Pat. No. 5,518,651 describes a method for the cleavage anddeprotection of insolubilized and protected oligonucleotides using analkyl amine, e.g. t-butylamine and methylamine. According to thispatent, the deprotection and cleavage of the oligonucleotides occurs atroom temperature and in less than about 90 min.

U.S. Pat. No. 5,738,829 describes a method for the cleavage anddeprotection of oligonucleotides from solid supports which involvesincubation of the immobilized oligonucleotides with gaseous ammonia orammonium hydroxide vapors. According to this patent, the method lendsitself to the use of supports such as microtiter plates that can be usedto perform up to 96 individual synthetic processes.

Glenn Research of Sterling Virginia offers phenoxyacetyl protected dA,4-isopropylphenoxylacetyl protected dG and acetyl protected dC which canbe used to prepare oligonucleotides. According to Glenn Research's website, these monomers can be used with sensitive labeling reagents suchas TAMRA, Cy5® and HEX since cleavage and deprotection can be carriedout in 2 hours at room temperature with ammonium hydroxide or 0.005 Mpotassium carbonate in anhydrous methanol. In addition, according tothis web site, it is possible to deprotect oligonucleotides containingacetyl protected dC monomers by treatment with ammoniumhydroxide/methylamine for 10 min at 65° C. or less.

We have now found that the use of nucleophilic amino compounds underpressure and high temperature is an effective way to deprotect dyelabeled oligos. The dye labeled oligos deprotected in this manner arefully deprotected and are of high quality. In addition, the process issimple and saves time, reducing the deblocking (processing) time fromapproximately 28 hours to 1 hour.

BRIEF SUMMARY OF THE INVENTION

The invention relates to a process for deblocking a detectably labeledoligonucleotide comprising contacting the blocked detectably labeledoligonucleotide with an effective amount of a nucleophilic aminocompound under conditions that result in the deblocking of theoligonucleotide, thereby giving the deblocked oligonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict ion-pair HPLC chromatograms showing the analysisof the dye labeled oligo (5′-FAM-GGT CCG ACC AGA TGG CGA AAG GCA AACGGA; SEQ ID NO:1) after deprotection with concentrated ammoniumhydroxide, RT, 24 hrs (FIG. 1A) or with gaseous ammonia (80 psi), 95°C., 45 min (FIG. 1B). Buffer A=5 mM TBAP in 20 mM NH₄HPO₄. BufferB=CH₃CN, gradient of 40% B to 60% B over 10 min, flow rate of 2 ml/min.

FIGS. 2A-2D depict ion-pair HPLC chromatograms showing the analysis ofmolecular beacon (5′-HEX-GCG ACG CCT GTC CTC CAA TTT GTC CTG GTC GTC GCDABCYL) after deprotection with concentrated ammonium hydroxide, 90° C.,75 min (FIG. 2A), concentrated ammonium hydroxide, RT, 24 hrs (FIG. 2B),concentrated ammonium hydroxide, RT, 40 hrs (FIG. 2C), or with gaseousammonia (80 psi), 95° C., 1 hr (FIG. 2D). Buffer A=5 mM TBAP in 20 mMNH₄HPO₄. Buffer B=CH₃CN, gradient of 40% B to 60% B over 10 min, flowrate of 2 ml/min.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a process for deblocking substantiallydetectably labeled oligonucleotides comprising contacting the blockeddetectably labeled oligonucleotide with an effective amount of adeblocking reagent such as a nucleophilic amino compound. Preferably,the deblocking reagent is gaseous at ambient temperature.

The present invention provides a number of advantages over conventionalmethods of deblocking oligonucleotides using aqueous ammonia, includingimproved quality (purity) of the deblocked oligonucleotide, higheryield, and shorter reaction times. Moreover, the amino compound may beremoved substantially by degassing (in the case of ammonia, methylamineand ethylamine), thus providing ease of recovery of the deblockedoligonucleotide. In the case of higher molecular weight amino compoundsthat are liquids at room temperature, the amino compound may be removedby washing the deblocked oligonucleotide with an organic solvent inwhich the oligonucleotide is poorly soluble, e.g. acetonitrile, diethylether and the like, to remove the amino compound. The deblockedoligonucleotide may then be resuspended directly in water, a buffer orother solution and used directly as a molecular biology reagent, e.g. asa diagnostic reagent in sequencing, PCR or as a probe. The buffer may bechosen to neutralize any residual nucleophilic amino compound (e.g.ammonia) that may be present. Particularly preferred buffers are theacetate, sulfate, hydrochloride, phosphate or free acid forms ofTris-(hydroxymethyl)amino-methane (TRIS®), although alternative buffersof the same approximate ionic strength and pKa as TRIS® may be used withequivalent results. Other preferred buffers are triethyl ammonium salts(e.g. the acetate salt). In addition to the buffer salts, cofactor saltssuch as those of potassium (preferably potassium chloride or potassiumacetate) and magnesium (preferably magnesium chloride or magnesiumacetate) may be included. Addition of one or more carbohydrates and/orsugars to the buffer solution and/or deblocking reaction mixtures mayalso be advantageous, to support enhanced stability of the product uponstorage. Preferred such carbohydrates or sugars include, but are notlimited to, sucrose, trehalose, and the like. Such carbohydrates and/orsugars are commercially available from a number of sources, includingSigma (St. Louis, Mo.).

The nucleophilic amino compound may be ammonia or ammonia vapors (e.g.obtained by heating a sealable chamber having a quantity of ammoniumhydroxide in the bottom), or a C₁₋₆ alkylamino compound. The alkyl groupmay be straight or branched chain. Examples of such alkylamino compoundsinclude methylamine, ethylamine, propylamine, isopropylamine,butylamine, sec-butylamine, pentylamine and hexylamine. When thenucleophilic amino compound is a liquid at ambient temperature, it maybe removed under vacuum with or without heating. In a preferredembodiment, the nucleophilic amino compound is at least saturated withwater vapor.

The label on the oligonucleotide may be any conventional label used fordetection of oligonucleotides, including, without limitation,fluorescent dyes chosen from the group consisting of xanthenes (e.g.,fluoresceins, eosins, erythrosins), rhodamines (e.g., Texas Red®),benzimidazoles, ethidiums, propidiums, anthracyclines, mithramycins,acridines, actinomycins, merocyanines, coumarins (e.g.,4-methyl-7-methoxycoumarin), pyrenes, chrysenes, stilbenes, anthracenes,naphthalenes (e.g., dansyl, 5-dimethylamino-1-naphthalenesulfonyl),salicylic acids, benz-2-oxa-1-diazoles (also known as benzofurans)(e.g., 4-amino-7-nitrobenz-2-oxa-1,3-diazole), indodicarbocyanines (e.g.Cy3® and Cy5®, available from Biological Detection Systems, Inc.),fluorescamine, and psoralen. See U.S. Pat. Nos. 4,997,928, 5,262,536,and EP 63,879. Useful forms of many of these dyes are commerciallyavailable. See also A. W. Wagner, Chapter 1, Applications ofFluorescence in the Biomedical Sciences, Taylor et al. (ed.), Alan R.Liss, New York (1986). Particular examples include6-(fluorescein-6-carboxamido)hexanoate (6-FAM), fluoresceinisothiocyanate (FITC), hexachlorofluorescein (HEX),tetrachlorofluorescein (TET),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (6-JOE), and BODIPY.In a preferred embodiment, the oligonucleotides is labeled with themolecular beacon technology according to Tyagi, S. and Kramer, E R.,Nature Biotechnology 14:303-308 (1996). One label that degrades in thepresence of ammonia is (6-tetramethylrhodamine (TAMRA).

The blocked, labeled oligonucleotides may be prepared by well knownmethods, e.g. the phosphoramidite, phosphotriester, phosphodiester,phosphite and H-phosphonate methods, each of which are generally knownin the field of molecular biology. For example, the b-cyanoethylphosphoramidite method is described in U.S. Pat. No. 4,458,066 issued toCaruthers, et al., entitled “Process for Preparing Polynucleotides,”which is incorporated herein by reference. See also E. Eckstein (ed.),Oligonucleotides and Analogs, A Practical Approach, IRL Press, Oxford(1991); GB 2,125,789; and U.S. Pat. Nos. 4,415,732, 4,739,044 and4,757,141. Such oligonucleotides may be DNA, RNA, mixture of DNA andRNA, derivatives of DNA and RNA and mixtures thereof. In the case ofRNA, base stable 2′-protecting groups are preferred. The blocked,labeled oligonucleotide may be free or immobilized on a solid phasewhich is also cleaved by the gaseous deblocking reagent. The deblockedoligonucleotide may then be recovered by washing the solid phase withwater or a buffer.

The blocking group is present on the exocyclic amino groups of A, G andC. Thymine does not require protection. Such blocking groups may be C₁₋₆alkanoyl (e.g. isobutyryl), aryloyl (benzoyl), phenoxyacetyl, C₁₋₆alkoxyacetyl, and dimethyl formamidine (on N⁶ of dA or N² of dG). Whenthe deblocking reagent is an alkylamine, it is preferred that the base Cbe blocked with an acetyl group. The blocking group for the phosphorousmay be a cyanoethyl group. All of these blocking groups are cleaved atthe same time by the deblocking reagent.

The deblocking reaction is preferably carried out in a sealable chamber(although an open chamber may be used in accordance with the invention)that can be heated. Such sealable chambers include screw cap vials, Parrbottles, and the like. The oligonucleotide synthesis and cleavage fromthe support may be carried out with a commercially available DNAsynthesizer, e.g. the ABI 380B DNA synthesizer, or other equipment thatis set up for high throughput synthesis on a multi well channel, e.g. a96 well plate (see, e.g., U.S. Pat. Nos. 5,472,672 and 5,529,756, andU.S. application Ser. No. 09/162,348, filed Sep. 28, 1998, which areincorporated herein by reference in their entireties).

The deblocking reagent is present in an amount effective to deblock theoligonucleotide. In general, the deblocking reagent is present in alarge excess compared to the oligonucleotide. In the case of ammonia,the sealable chamber may be charged with about 20 to 200 psi of ammonia,most preferably, about 80 psi. Optimal amounts of the liquid alkylaminocompounds may be determined with no more than routine experimentation.

The deblocking reaction is carried out at a temperature of about roomtemperature to about 150° C. Most preferably, when the deblockingreagent is ammonia, the reaction is carried out at about 95° C.

The deblocking reaction is carried out for about 1 min to about 2 hrs.More preferably, the reaction is carried out for about 1 min to about 1hr. When the deblocking reagent is ammonia, it is preferred that thereaction be carried out for about 45 min.

By “substantially deblocked” is intended to mean that the blockedoligonucleotide is not detectable, e.g. by ion-pair HPLC, capillaryelectrophoresis or mass spectrometry, after the deblocking reactionaccording to the present invention.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in molecular biology and chemistry, particularlyoligonucleotide synthesis, which are obvious to those skilled in the artin view of the present disclosure are within the spirit and scope of theinvention.

EXAMPLES

An example of deblocking of a fluorescein (FAM) labeled oligo understandard conditions (room temperature and 24 hrs) and also gas phase (45min, 80 psi, 95° C.) is attached (FIGS. 1A and 1B). As can be seen fromthe chromatogram, the peak for the standard deblocking is broaderindicating that there is an incomplete deprotection. However, thechromatogram deblocking with ammonia shows a much sharper peak which isindicative of complete deprotection and no degradation.

In another example an oligonucleotide labeled with hexachlorofluorescein(HEX) and DABCYL (molecular beacon) was deblocked under variousconditions (FIGS. 2A-2D). Standard deblocking conditions (95° C.,concentrated ammonia, 75 min, FIG. 2A) as well as room temperaturetreatment over 24 hours (FIG. 2B and FIG. 2C) show formation of a newpeak which may be attributed to degradation of the dye. As can be seenfrom the chromatographs (FIG. 2D) deblocking under gas phase givesbetter quality oligonucleotides.

Experimental Procedure:

Place the dye-oligo-CPG (obtained from phosphoramidite based automatedsynthesis) in a high pressure reactor. Charge the reactor with gaseous,ammonia saturated with water vapor (80 psi pressure). Heat the sealedreactor to 95° C. for 45 min. Release the pressure and cool the CPG toroom temperature. Elute the oligonucleotide from the CPG with water(˜300 μl is sufficient for 50-200 nmol scale synthesis) and proceed toanalysis and purification.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those in the artto which the invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference in theirentirety.

1. A process for deblocking substantially a blocked, detectably labeledoligonucleotide comprising contacting the blocked detectably labeledoligonucleotide with an effective amount of a nucleophilic aminocompound under conditions that result in the deblocking of theoligonucleotide, thereby giving the substantially deblockedoligonucleotide.
 2. The process of claim 1, wherein said detectablelabel is a fluorescent label.
 3. The process of claim 2, wherein saiddetectable label is hexachlorofluorescein.
 4. The process of claim 2,wherein said detectable label is DABCYL.
 5. The process of claim 1,wherein said nucleophilic amino compound is ammonia.
 6. The process ofclaim 5, wherein said ammonia is present at a psi of about 20 to
 200. 7.The process of claim 5, wherein said ammonia is present at a psi ofabout
 80. 8. The process of claim 1, wherein said nucleophilic aminocompound is ammonia vapors.
 9. The process of claim 1, wherein saidnucleophilic amino compound is a C₁₆ alkylamine.
 10. The process ofclaim 1, further comprising dissolving the substantially deblockedoligonucleotide in a buffer.
 11. The process of claim 1, wherein saidconditions comprise carrying the process at about room temperature toabout 150° C.
 12. The process of claim 1, wherein said conditionscomprise carrying the process at about 95° C. 13-14. (canceled)
 15. Theprocess of claim 1, wherein said substantially blocked, detectablylabeled oligonucleotide is immobilized on a solid phase.
 16. The processof claim 15, wherein said substantially deblocked oligonucleotide isreleased from said solid phase under said conditions.
 17. The process ofclaim 16, wherein said substantially deblocked oligonucleotide isrecovered by washing said solid phase with water or a buffer.
 18. Aprocess for deblocking substantially a blocked, detectably labeledoligonucleotide comprising contacting the blocked detectably labeledoligonucleotide with an effective amount of ammonia saturated with watervapor at about 80 psi, 95° C. for about 45 min, thereby giving thesubstantially deblocked oligonucleotide. 19-21. (canceled)
 22. Acomposition comprising a blocked, detectably labeled oligonucleotide andan effective amount of a nucleophilic amino compound sufficient todeblock substantially said oligonucleotide.
 23. The composition of claim22, wherein said detectable label is a fluorescent label.
 24. Thecomposition of claim 22, wherein said nucleophilic amino compound isammonia.
 25. The composition of claim 22, wherein said nucleophilicamino 15 compound is ammonia vapors.